The present invention is concerned with certain improvements in test reagents for screen testing and the bioassay of von Willebrand's factor and platelet aggregating factor in human and animal plasmas as well as a new procedure therefore.
The chemistry and makeup of human and animal blood plasma has long been a topic of study in medical science. The coagulation of blood is a subject of much interest and complexity. One of the blood plasma proteins that is needed by the body for the prevention of hemorrhage is variously referred to as von Willebrand's factor (vWF), platelet aggregating factor (PAF), factor VIII:vWF, ristocetin cofactor, or as vWF/PAF. This plasma factor is reduced or absent in a bleeder disease, so-called von Willebrand's disease or vascular hemophilia, which occurs in both man and animals (e.g. dog and pig). This plasma factor is also altered in other disease states, and may be important in the development of atherosclerosis by recruiting platelets to the site of pre-atherosclerotic vascular injury.
Currently, there are two main procedures used in clinical laboratory diagnosis and blood plasma analysis for the determination of this plasma factor, sometimes referred to hereinafter as vWF/PAF. Both of these procedures utilize the following reagents:
(1) citrated plasma or other body fluid being tested for amount of vWF/PAF;
(2) a suspension of blood platelets for the determination of the presence or absence of platelet aggregation, and where present, its rate;
(3) a cofactor ristocetin which for certain plasma-platelet preparations (as human plasma with human platelets) is required in addition to vWF/PAF for platelet aggregation.
Ristocetin has been described by Howard et al (Ristocetin--a new tool in the investigation of platelet aggregation. Thromb. Diath. Haemorrh. 26:362-69, 1971) as capable of precipitating fibrinogen from plasma in vitro as well as producing platelet aggregation. It is a substance of uncertain chemical structure isolated from Nocardia lurida and is available in lyophilized form from Abbott Laboratories, Chicago, Ill. However, Fehlner et al have recently studied the structure of ristocetin and found that upon hydrolysis, a number of amino acids of unusual structure are obtained (See Proc. Nat. Acad. Sci., Vol. 69, No. 9, pp. 2420-2421, September 1972).
According to Weiss et al (Quantitative assay of a plasma factor deficient in von Willebrand's disease that is necessary for platelet aggregation. J. Clin. Invest. 52:2708-16, 1973) ristocetin is used in an aggregometer in order to quantitatively determine the von Willebrand's platelet aggregating factor. The "end point" employed according to this method is a tracing based on changes in light transmission through a platelet suspension upon the addition of vWF/PAF. Unfortunately this method is technically laborious and the number of samples which may be run in a half-day is very limited. Furthermore, tracings take time to record and require expensive instrumentation. The method further requires the use of washed platelet suspensions which are time consuming to prepare and many preparations are discarded due to spontaneous aggregation.
The second commonly used diagnostic method measures the macroscopic platelet aggregation time (i.e. determination of the rate of aggregation of a platelet suspension). Sarji et al (Nature of von Willebrand's factor: a new assay and a specific inhibitor. Proc. Nat. Acad. Sci. USA 71:2937-41, 1974 the entire contents of which are incorporated herein by reference) describe the use of ristocetin in this simplified test procedure utilizing the macroscopic platelet aggregation time for the determination of von Willebrand's factor. Aggregation times commonly are in the neighborhood of 30 seconds or less and the end point is observed visually. The number of samples which can be run in a half-day greatly exceeds that of the aggregometry method of Weiss et al. Nonetheless, this method also suffers the technical problem of laborious preparation of platelets.
More recently, it has been discovered that platelets fixed with (para)formaldehyde could be used as readily as fresh platelets resulting in the advantage that a batch of fixed platelets could be prepared and preserved for use for a period of one to two months (see Allain et al--Platelets fixed with paraformaldehyde: a new reagent for assay of von Willebrand's factor and platelet aggregating factor. J. Lab. Clin. Med. 85:318-28, 1975 which is incorporated herein by reference). The fixed platelets when employed in the macroscopic test of Sarji et al have been found to result in a shorter aggregation time using ristocetin as a cofactor than when fresh platelets are employed (i.e. 10-20 seconds as compared with 30 seconds). The fixed platelets have been shown to be equally as useful in the assay of platelet aggregating factor (PAF) in animal plasma which clumps human platelets. Platelet aggregating factor is an activity of the same plasma protein as von Willebrand factor of human plasma detected by ristocetin-induced aggregation of human platelets. The use of fixed platelets resulted in the additional advantage that they were insensitive to other platelet aggregating agents which might be present in the plasma sample being tested, such as thrombin or adenosine diphosphate or thromboxanes, prostaglandin derivatives.
However, two exceptions were encountered regarding the use of ristocetin cofactor reagent in the test for von Willebrand's factor and platelet aggregating factor. The first related to the ability of types of animal plasmas (e.g. pig and cow) to aggregate fresh or fixed human platelets without ristocetin. Secondly, certain plasmas such as dog would not aggregate either human or dog platelets if ristocetin were present, and thus no assay could be readily obtained on these plasmas.
Furthermore, it was found that both patients with von Willebrand's disease as well as with other diseases may acquire an inhibitor to the vWF/PAF, i.e. a plasma gamma globulin antibody. Both the procedures of Weiss et al and Sarji et al may be employed to detect presence of the inhibitor and by serially diluting the patients' plasma, the titer of the inhibitor can be determined with these tests.
Accordingly, it is the principle object of the present invention to provide a test reagent for the bioassay of von Willebrand's factor and platelet aggregating factor in blood plasmas which meets the requirements set forth above and obviates difficulties and disadvantages encountered with prior art test reagents.
A further specific object of the present invention is to provide and modify prior test reagent compositions thereby providing a more effective and accurate means of determining the von Willebrand's factor and platelet aggregating factor in human and animal blood plasmas.
Still yet a further object of the present invention is to provide a procedure for the accurate determination of von Willebrand's factor and platelet aggregating factor in human and animal blood plasmas.
Yet another object of the present invention is to provide for the inclusion of dried platelets as a reagent for the testing of vWF/PAF in human and animal blood plasmas.
These and other objects will be apparent from the description of the invention which follows hereinbelow.